Current approaches to transfer genes in vivo employ either recombinant viral vectors or non-viral delivery systems. We are engaged in studying the molecular biology of the human parvovirus adeno-associated virus (AAV) with the intent to using this virus as a platform for developing a novel, safe, and efficient delivery system for human gene therapy. Our research pioneered the use of? recombinant AAV (rAAV) as a gene delivery system for central nervous system, and muscle cells demonstrating vector expression for over 1.5 years without immune consequences or vector toxicity. While promising, these studies uncovered rate limiting steps involved in AAV vector transduction; namely receptor mediated entry, and conversion of singled-stranded viral genomes to double-stranded expressing templates (i.e. second-strand synthesis). In addition to this rate-limiting step, the finite packaging capacity of this virus (4.5kb) has restricted the use of this vector to small genes or cDNAs. To advance the prospects of efficient AAV gene delivery, vectors sufficient to carry larger genes must be developed. In addition, virions that specifically and efficiently target defined cell types will be required for clinical application. The focus of this grant will be to address these issues. Three approaches will be analyzed in this proposal to overcome AAV?s problem of viral entry, inefficient second-strand synthesis, and packaging constraints. We have generated exciting new mouse data supporting differential infectivity in type I vs. type II muscle using traditional AAV type II vectors, as well as increased transduction when using other AAV serotype specific vectors. These results emphasize the importance of receptor mediated AAV entry. Mapping and characterization of other AAV serotype capsid domains required for entry will be explored to facilitate more infectious vectors. AAV type II as well as serotype specific vectors 1-5 will be tested in the Chapel Hill canine model for efficient FIX gene delivery in an effort to extend current mouse studies in a large animal model. We have now developed a method for generating AAV vectors carrying duplex viral genomes. These reagents will be characterized for efficient transgene expression after vector delivery in an effort to study rate-limiting steps involved in second-strand synthesis and vector gene expression in vivo. Finally, efforts to engineer vectors that carry twice the packaging capacity of wild type AAV will be explored by using split gene vectors that rely on hetero-dimer concatemers after vector infection. The long term objective is to develop novel delivery systems that exploit the advantages of AAV viral infectivity without the disadvantages of inefficient viral entry, rate limiting steps involved in second-strand synthesis, or packaging constraints.